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        <title>Automated LRE Window Selection</title>
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        <h1 align="center">Automated LRE Window Selection</h1>
		<p>LRE window selection begins with assigning a &quot;start cycle&quot;, which 
		defines the bottom of the LRE window and corresponds to the earliest 
		cycle within the LRE window. The LRE window is then expanded by adding one 
		cycle at a time to the top of the window until a limit is reached. Effective automation of this 
		process is crucial to the reliability of 
		the LRE Analyzer, and was one of the most challenging aspects of its development. </p>
		<p><b>Optical read precision is critical for start cycle selection </b></p>
		<p>Although a primary objective was to maximize the size of the LRE window 
		by placing the start cycle as early in a profile as possible, optical
		<a href="../glossary/glossary.html#Read_Precision">read precision</a> 
		became a major limitation. This is due to the fact that the accuracy of
		<a href="../glossary/glossary.html#Cycle_Efficiency">cycle efficiency</a> 
		determination can be dramatically compromised when reaction fluorescence is below the 
		lower limit of the instrument&#39;s optical capacity. Large differences in 
		this lower limit between different instruments, combined with the 
		arbitrary nature of the fluorescence units used in real-time PCR, 
		presented major challenges. </p>
		<p>This prompted a default implementation based on a simple, albeit 
		suboptimal method of designating the start cycle as the first cycle 
		below <a href="../glossary/glossary.html#C1/2">C<sub>1/2</sub></a>. 
		However, although this approach can be reasonably reliable under a 
		number of <a href="../glossary/glossary.html#Reaction_Setup">reaction setups</a>, an alternative method was 
		developed that allows the lower limit of the LRE window to be manually specified. 
		Based on entry of a&nbsp; &quot;minimum F<sub>C</sub>&quot;, the start cycle is 
		set to the cycle following the first cycle that produces a F<sub>C</sub> greater than this 
		minimum (i.e. the cycle from which the E<sub>C</sub> denominator is 
		taken). The &quot;LRE Window Selection Parameters&quot; panel within the
		<a href="../editor_panel/editor_panel_overview.html">Profile Editor</a> 
		window, allows the minimal F<sub>C</sub> and F<sub>0</sub> threshold 
		(see below) to 
		be adjusted manually.</p>
		<p align="center"><b>Default Settings<br>
		</b>
		<img border="0" src="images/window_parameters1.gif"></p>
		<p><b>Selecting a Minimum F<sub>C</sub></b></p>
		<p>During early implementation of the LRE window parameters, it became 
		apparent that a method for assessing the overall quantitative precision 
		could be useful. The approach taken was based on the variance of target 
		quantities generated by technical replicates; that is, the CV produced by replicate profiles. Referred to as the &quot;Av Repl CV&quot; 
		(and assuming that replicate profiles are present within the 
		database) averaging the quantitative variances from all replicate 
		reaction sets provides such a general assessment. This not only proved to 
		useful for selecting an optimal minimum F<sub>C</sub>, but also for 
		assessing the overall performance of an assay. Although beyond the scope 
		of this discussion, this has revealed, among other things, large 
		differences in the instrument performance, due primarily to differences 
		in the optical precision they produce. </p>
		<p>A simple and generally effective method is thus to lower the minimum 
		F<sub>C</sub> until the average replicate CV reaches a minimum. </p>
		<p align="center"><b>Manual selection of Min F<sub>C</sub><br>
		</b>
		<img border="0" src="images/window_parameters2.gif"></p>
		<p align="left">In this example, the overall quantitative variance has 
		been reduced from 4.7% to 4.5%.</p>
		<p><b>Defining the top of the LRE window via the F<sub>0</sub> threshold</b></p>
		<p>A major source of quantitative error discovered during early attempts 
		to apply sigmoidal mathematics to PCR amplification, are distortions 
		within the upper region of a profile<sup><a href="#1.">1</a></sup>. For 
		example, a common form, referred to as &quot;plateau drift&quot;, is characterized 
		by a continued increase in amplicon DNA beyond that predicted 
		by the LRE model. In order to maximize quantitative accuracy, it is 
		essential to exclude such aberrant cycles from the analysis<sup><a href="#1.">1</a></sup>. 
		The recursive nature of LRE analysis provided a relatively simple method for 
		identifying aberrant cycles, which are apparent in both the F<sub>C</sub> 
		and LRE plots:</p>
		<p align="center"><b>An example of plateau drift<br>
		</b>
		<img border="0" src="images/plateau_drift.gif" width="355" height="312"></p>
		<p align="left">Particularly in the LRE plot, aberrant cycles are 
		evident as they diverge from the LRE line, which in the case of plateau 
		drifting produces points that progressively drift above the LRE line. An 
		objective method for defining the top of the LRE window came from 
		comparing the <a href="../glossary/glossary.html#Cycle_F0">cycle F<sub>0</sub></a> 
		of the cycle immediately above the LRE window to the
		average F<sub>0</sub> values generated by the cycles within the LRE window (<a href="../glossary/glossary.html#Average_F0">average F<sub>0</sub></a>). 
		If the difference is below a specified value, defined as the
		<a href="../glossary/glossary.html#F0_Threshold">F<sub>0</sub> threshold</a>, 
		the LRE window is expanded to include this next cycle and LRE analysis 
		repeated. This process is 
		continued until a cycle that exceeds the F<sub>0</sub> threshold is 
		encountered. As shown above in the LRE Window Selection Parameters 
		panel, the default value is 6%.</p>
		<p align="left">The Tabular Summary provides a numerical perspective of the 
		process, where &quot;%Av. Fo&quot; refers to the difference between a
		<a href="../glossary/glossary.html#Cycle_F0">cycle&#39;s F<sub>0</sub></a> 
		and the <a href="../glossary/glossary.html#Average_F0">average F<sub>0</sub></a>, 
		expressed as a 
		percentage:</p>
		<p align="center"><b>Tabular Summary: 6% F<sub>0</sub> threshold<br>
		</b>
		<img border="0" src="images/table_plateau_drift.gif" width="258" height="207"></p>
		<p>In this example, cycle 29 generated a 6.84% difference and thus LRE 
		window expansion was terminated at cycle 28 based on the 6% default that 
		was applied in this example, which been found to be generally effective. It should be 
		noted, however, that increasing the threshold much above 7% can lead to 
		susceptibility to another form of distortion, referred to as &quot;profile 
		collapse&quot;:</p>
		<p align="center"><b>An example of profile collapse<br>
		</b>
		<img border="0" src="images/plateau_collaspe.gif" width="357" height="312"></p>
		<p>In this case the aberrant cycles fall below the LRE line. Although a detailed overview is beyond the 
		scope of this discussion, when such cycles are included into the LRE 
		analysis, E<sub>max</sub> is overestimated , which in turn generates an 
		underestimate of target quantity. </p>
		<p>See also:<br>
		<a href="manual_window_selection.html">Manual Window Selection</a> </p>
		<p><a name="1.">1.</a> Rutledge, RG (2004) Sigmoidal curve-fitting 
		redefines quantitative real-time PCR with the prospective of developing 
		automated high-throughput applications. Nucleic Acids Research 32: e178.</p>
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